Worm Breeder's Gazette 13(2): 97 (February 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Do P granules contain messenger RNA?

Geraldine Seydoux, Andy Fire. Carnegie Institution, Baltimore, MD 21210.

We have analyzed the distribution of poly-A+ mRNAs in embryos by in situ hybridization using a digoxigenin-labeled oligo-dT probe and a rhodamine-labeled anti-digoxigenin antibody. The cytoplasm of all cells at all stages hybridizes strongly with the oligo-dT probe. In the P lineage (P(0)-P(4)), the oligo-dT staining pattern forms cytoplasmic granules. In P(0), P(1), and P(2), these granules are preferentially associated with the posterior half of the cell cortex prior to each division. In P(3) and P(4), the granules coalesce and become peri-nuclear. This dynamic pattern of localization is identical to the pattern previously reported for P granules(1). To determine if the oligo-dT-hybridizing granules were indeed P granules, we performed double label in situ experiments with P granules labeled with an FITC-conjugated antibody (OI C1D4 ,thanks to Susan Strome). In these experiments, we found that the majority of the oligo-dT-hybridizing granules co-localized with P granules. These results suggest that poly-A+ RNA is present in P granules.

As a control for specificity of the in situ hybridization protocol, we have used an oligo-dA probe, which gives no staining. In addition, pre-treatment of embryos with RNAse A or RNAse ONE eliminates staining with oligo-dT.

To characterize further the P granule-associated RNAs, we have determined the localization of SL1 -containingRNAs, using a probe complementary to the 22 nucleotides of SL1 that are trans-spliced upstream of many RNAs(2). Like the oligo-dT probe, this anti-SL1 probe hybridizes to the cytoplasm of all cells and to cytoplasmic granules in the P cells (presumably P granules). In contrast, an oligo complementary to the 3' region of the SL1 precursor does not show any staining. We have also used a monoclonal antibody (K121, thanks to Joe Gall) that recognizes the trimethylguanosine cap found at the 5' end of many small RNAs, including SL13 .Like the oligo-dT and anti-SL1 probes, this antibody recognizes putative P granules. (In addition, this antibody also binds to 1-2 foci in somatic cell nuclei.) Together, these results suggest that SL1 -containingRNAs are present in P granules.

What is the identity of the RNAs associated with P granules? Because these RNAs are detected as early as the one-cell stage, we presume that they are maternal RNAs. As reported in the last WBG (Vol 13.1, p25 ),we have determined the localization of 6 maternal mRNAs by in situs ( cey-1 , cey-2 , glp-1 , skn-1 , eIF4 A, HSP70 a).Although all of these RNAs are present in the P lineage, none appear to be preferentially associated with P granules in the 1 to 4-cell stage. Later, when the P granules start to coalesce around the nucleus in P3 and P4 ,these maternal RNAs often also adopt this peri-nuclear localization, but the significance of this late association remains unclear. We are investigating the possibility that P granule-associated RNAs correspond to a yet unidentified class of maternal mRNAs.

1. Strome and Wood, 1982. PNAS 79,1558-1562.

2. Krause and Hirsh, 1987. Cell 49, 753-761.

Bektesh et al., 1988. Genes and Development 2,1277-1283.

3. Liou and Blumenthal 1990. Mol. Cell Biol. 10, 1764-1768.

Van Doren and Hirsh, 1990. Mol. Cell Biol. 10,1769-1772.